Method of detecting risk of type II diabetes based on mutations found in carboxypeptidase E

ABSTRACT

The invention is directed to methods for diagnosing Type II diabetes or the risk for developing Type II diabetes by detecting alterations in expression, sequence, or function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods for preventing or treating Type II diabetes by modulating the levels, altering the sequence, or controlling the function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods for identifying agents that modulate the levels or affect the function of carboxypeptidase E nucleic acid or protein. The invention is also directed to methods using the agents to treat or diagnose Type II diabetes. The invention is also directed to animal models of Type II diabetes using the carboxypeptidase E gene. The invention is also directed to compositions based on carboxypeptidase E nucleic acid or protein useful for treating or diagnosing diabetes, identifying compounds for treating or diagnosing diabetes, and developing animal models of diabetes.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. application Ser. No.09/233,989, filed on Jan. 19, 1999 which claims the benefit of U.S.Provisional Application No. 60/105,102, filed Oct. 21, 1998, which ishereby incorporated in its entirety by reference.

FIELD OF THE INVENTION

[0002] The invention is directed to methods for diagnosing Type IIdiabetes or the risk for developing Type II diabetes by detectingalterations in expression, sequence, or function of carboxypeptidase Enucleic acid or protein. The invention is also directed to methods forpreventing or treating Type II diabetes by modulating the levels,altering the sequence, or controlling the function of carboxypeptidase Enucleic acid or protein. The invention is also directed to methods foridentifying agents that modulate the levels or affect the function ofcarboxypeptidase E nucleic acid or protein. The invention is alsodirected to methods using the agents to treat or diagnose Type IIdiabetes. The invention is also directed to animal models of Type IIdiabetes using the carboxypeptidase E gene. The invention is alsodirected to compositions based on carboxypeptidase E nucleic acid orprotein useful for treating or diagnosing diabetes, identifyingcompounds for treating or diagnosing diabetes, and developing models ofdiabetes.

BACKGROUND OF THE INVENTION

[0003] Carboxypeptidase E

[0004] Carboxypeptidase E (CPE), known also as carboxypeptidase H andenkephalin convertase, is involved in the processing of variousbioactive peptides including peptide hormones and neurotransmitters(Fricker, in Peptide Biosynthesis and Processing) Fricker, ed. (pages199-230 CRC Press, Boca Raton, Fla.) (1991). Many peptide hormones andneurotransmitters are initially produced as precursors that areenzymatically processed into bioactive peptides (Fricker J. CellBiochem. 38:279-289 (1988)). Initially, endopeptidases cleave theprohormone precursor at multiple basic amino acid cleavage sites(Varlamov et al. J. Biochem. 271:13981-13986) (1996)). Then acarboxypeptidase removes the basic amino acids from the C terminus ofthe peptide to generate either the bioactive product or a precursor toform the C-terminal amide group. This process is important for theproduction of bioactive peptides in many tissues.

[0005] CPE is present in many tissues where peptide biosynthesis occursincluding brain, pituitary, and adrenal medulla (Fricker, J. Cell.Biochem., cited above). The activity is localized to secretory granuleswhere CPE exists in membrane and soluble forms (Manser et al. Biochem.J. 267:517-525 (1990)). CPE does not appear to contain atransmembrane-spanning helical region, which suggests that CPE ismembrane bound through another mechanism. A recent study has shown thatthe C-terminal region of CPE particularly the C-terminal 14 amino acidsare required for membrane binding (Varlamov et al. J. Biol. Chem.271:6077-6083 (1996)). Using deletion mutation and fusion proteinanalysis to study membrane binding and targeting, the authors concludedthat there were three separate functions within the C-terminal region ofCPE. The 51 C-terminal amino acids appear to direct the sorting to themembrane. Another important region, located 23-33 amino acids from theC-terminus appear to be required for proper folding in that proteinlacking this region is neither active nor secreted. A third domain,located within the predicted amphiphilic helix of the C-terminal 14residues was involved with a binding of CPE to membranes.

[0006] A high degree of conservation of the C-terminal region among CPEfrom different species has been shown. The last exon which encodes theC-terminal 32 amino acids is a hundred percent identical in human, rat,mouse, and bovine CPE and contains only 4 conservative substitutions inangler fish CPE (Varlamov, J. Biochem. 271, cited above).

[0007] Within secretory granules, CPE has been shown to be present inseveral forms having different solubility. Different forms of CPE havebeen purified to apparent homogeneity (Supattapone et al. J. Neurochem.42:1017 1984); and (Fricker et al. J. Biol. Chem. 258:10950 (1983)).Soluble and membrane associated forms have similar enzymatic andphysical properties (Fricker, J. Cell. Biochem., cited above). Bothforms have the same amino acid sequence at the N-terminal region(Fricker et al. Nature 323:461 (1986)). It has thus been suggested thatdifferences between soluble and membrane forms may be the result ofpost-translational modifications of a single precursor protein (FrickerJ. Cell. Biochem., cited above). It has been shown that membrane andsoluble forms of CPE are synthesized in the rough endoplasmic reticulumand apparently derived from a single mRNA species by post-translationalprocessing. Synthesis of translation products from human CPE mRNA in areticulocyte lysate in the presence of microsomal membranes produced 3processed forms of CPE also showing differences in glycosylation (Manseret al., cited above).

[0008] Non-insulin-dependent Diabetes Mellitus

[0009] Diabetes mellitus is among the most common of all metabolicdisorders, affecting up to 11% of the population by age 70. Type Idiabetes (insulin-dependent diabetes mellitus or IDDM) represents about5 to 10% of this group and is the result of progressive autoimmunedestruction of the pancreatic β-cells with subsequent insulindeficiency.

[0010] Type II diabetes (non-insulin dependent diabetes mellitus, ortype II diabetes) represents 90-95% of the affected population, morethan 100 million people worldwide (King et al. (1988) Wld. Hlth.Statist. Quart. 41:190-196; Harris et al. (1992) Diabetes Care15:815-819), and is associated with peripheral insulin resistance,elevated hepatic glucose production, and inappropriate insulin secretion(DeFronzo, R. A. (1988) Diabetes 37:667-687). Family studies point to amajor genetic component (Newman et al. (1987) Diabetologia 30:763-768;Köbberling, J. (1971) Diabetologia 7:46-49; Cook, J. T. E. (1994)Diabetologia 37:1231-1240). However, few susceptibility genes have beenidentified.

[0011] Familial predisposition for obesity is the major phenotypic riskfactor associated with development of type II diabetes in humans. Thisobesity is usually accompanied by the development of insulin resistance(Naggert et al. Nature: Genetics 10: 135-141 (1995)).

[0012] In mice, six different loci on five different chromosomes producethe obesity-diabetes syndrome. These mutations affect not only obesityand insulin resistance but also other neuroendocrine disturbances. Oneof these mutations (fat/fat) is associated with a lesion in the CPEgene. The fat mutation maps to mouse chromosome 8 close to the gene forCPE. It was first shown that in extracts of fat/fat pancreatic isletsand pituitaries, proinsulin processing was severely reduced. This wasassociated with a ser202pro mutation in the CPE coding region. Thismutation was shown to abolish enzymatic activity in vitro. Thus, thismutation was proposed to demonstrate an obesity-diabetes syndrome causedby a defect in a prohormone processing pathway, i.e., in CPE (Naggert etal. cited above). The importance of this mutation in CPE function inmice was further investigated by studying the effects on activity,amount, and properties of CPE with ser to pro, ala, gly, or phesubstitutions at amino acid 202. In an in vitro system, phe and promutants were enzymatically inactive, could not bind to a substrate, andwere not secreted. Ala or gly mutants, however, exhibited normalenzymatic activities. In a mouse pituitary derived cell line, pro andphe mutants were not secreted. Further, they were degraded withinseveral hours. The analysis of CPE from pituitary cells derived fromfat/fat mice showed that the natural pro mutant produced in these cellswas not secreted but was degraded. These results provided furthersupport for the hypothesis that fat/fat mice are defective in CPEactivity because of the ser to pro substitution at amino acid 202. Asubsequent study examined CPE activity and peptide processing in severaltissues of fat/fat mice. The report found that there is no active CPE inthese mice. It was concluded therefore that the ser to pro mutationcauses the enzyme to be completely inactive. It was also concluded thatthe absence of active CPE causes a large decrease in the levels of fullyprocessed peptides, such as the enkephalins (Fricker et al. J. Biol.Chem. 271:30619-30624 (1996)). These results were consistent with thosefound for proinsulin and proneurotensin in the fat/fat mouse.Accordingly, the reference concluded that a deficiency of CPE in thefat/fat mouse leads to a dramatic accumulation of peptides withC-terminal basic residues, and a decrease in the levels of correctlyprocessed peptides. Although the authors proposed several mechanisms bywhich CPE acts on prohormones (for example, by being required forendopeptidase activity), the actual mechanism was not elucidated.

[0013] A recent report addressed the question of whether proinsulintargeting to secretory granules is impaired in fat/fat mice. The reportshowed that CPE is not essential for sorting of proinsulin to thesegranules (Irminger et al. J. Biol. Chem. 272:27532-27534 (1997)).

[0014] Although defects in loci encoding proinsulin conversion enzymeshave been postulated as a mechanism for producing hyperproinsulinaemiain humans, clinical cases demonstrating genetic defects in this pathwayin humans have not appeared definitively in the literature. One reportcited a severely obese Caucasian female patient who exhibited a possibledefect in the prohormone convertase 1-catalyzed conversion of proinsulin(Naggert et al., cited above).

[0015] Recently, the question of whether CPE plays a role in thepathogenesis of type II diabetes in humans was addressed (Utsunomiya, etal. Diabetologia 41:701-705 (1998)). Insulin is synthesized in thepancreatic β cell as a prohormone that is converted to insulin andC-peptide by the action of prohormone convertase II, prohormoneconvertase III, and CPE. In type II diabetes, the proinsulin leveland/or proinsulin: insulin ratio is increased. It was thus consideredthat mutations in these enzymes could contribute to the development oftype II diabetes. Further, the identification of a mutation in a CPEgene of the fat/fat mouse that is associated with hyperproinsulinemiaand late onset obesity-diabetes suggested the possibility that amutation in CPE might be involved in the development of these syndromesin humans. Thus, the CPE gene was screened for mutations in a group ofhuman subjects with type II diabetes and obesity. 269 subjects with typeII diabetes, 28 non-diabetic obese subjects, and 104 non-obese andnon-diabetic controls were studied. No correlation could be made betweena CPE gene nucleotide substitution and type II diabetes or obesity. Theauthors noted that although the relationship between the loss of CPEactivity and obesity-diabetes was not clear, the loss of CPE activitydid cause defects in the processing of prohormone neuropeptidesassociated with controlling satiety. However, the authors concluded thatnone of the nucleotide substitutions were associated with NIDDM orobesity and that genetic variation in the CPE gene does not appear toplay a major role in the pathogenesis of NIDDM or obesity in humans.

[0016] Accordingly, there is still a need to identify genetic factorsthat are important in developing type II diabetes. It is specificallyimportant to determine if the CPE gene could be useful for treating ordiagnosing type II diabetes in humans.

SUMMARY OF THE INVENTION

[0017] A general object of the invention is to identify polynucleotidesand polypeptides associated with the development of or risk fordeveloping Type II diabetes.

[0018] A further general object of the invention is to use thesepolypeptides and polynucleotides for treatment or diagnosis of Type IIdiabetes.

[0019] A further general object of the invention is to use thesepolypeptides and polynucleotides to identify compounds modulating theexpression or function of the polypeptides or polynucleotides.

[0020] A further general object of the invention is to use thesecompounds to modulate or otherwise interact with the polypeptides andpolynucleotides associated with Type II diabetes for the treatment anddiagnosis of the disorder.

[0021] A more specific object of the invention is to exploit therelationship between altered levels of expression or mutation in the CPEgene and the development of or risk of developing type II diabetes inhumans. Thus, it is a further specific object of the invention to useCPE polypeptides and polynucleotides for treatment and diagnosis of typeII diabetes and for identifying compounds that can modulate expressionor function of the polypeptides or polynucleotides and are thus usefulfor treatment and diagnosis of type H diabetes.

[0022] The invention is based on the inventors' discovery that mutationsin the carboxypeptidase E gene correlate with Type II diabetes inhumans.

[0023] Accordingly, the invention is directed to CPE polynucleotides andpolypeptides that are associated with the development of or risk fordeveloping Type II diabetes. In a specific disclosed embodiment, theinvention encompasses a coding mutation, arg→trp 283, corresponding to ac→t nucleotide change at this position.

[0024] The invention is also directed to using CPE polypeptides andpolynucleotides for treatment and diagnosis of Type II diabetes. Thepolypeptides and polynucleotides serve as both targets and reagents fortreatment and diagnosis.

[0025] The invention is also directed to using the polynucleotides andpolypeptides to identify compounds that are useful in the treatment anddiagnosis of Type II diabetes. The compounds can act as agonists orantagonists of CPE expression or function. The polynucleotides andpolypeptides serve as both a target to identify compounds and maythemselves provide a source for derivative compounds that can act as anagonist or antagonist of CPE expression or function.

[0026] The invention is further directed to using these compounds totreat and diagnose Type II diabetes.

[0027] Specifically, the invention is directed to methods for detectingan abnormal level of the CPE gene or gene product, a mutation in thegene or gene product, or otherwise abnormal gene or gene product, incells or tissues of individuals having type II diabetes or the risk ofdeveloping type II diabetes or in cell or animal models of thedisorders.

[0028] The invention is thus directed to methods for screening for TypeII diabetes or the risk of developing Type II diabetes by detecting theCPE gene or gene product. Abnormal levels, mutation, or otherabnormality allows the diagnosis of Type II diabetes or risk ofdeveloping Type II diabetes. In one embodiment, the invention isdirected to monitoring treatment outcome in a patient, in clinicaltrials, or in animal models, by detecting the CPE gene or gene productfor abnormal levels, mutation, or other abnormality.

[0029] The invention is thus also directed to methods for treatingindividuals with type II diabetes, or the risk of developing type IIdiabetes, using the abnormal levels, mutation, or other abnormality inthe CPE gene or gene product as a reagent or target for treatment. Inone embodiment, methods are directed to treating cells, tissues, oranimal models associated with the disorder using the CPE gene or geneproduct as a reagent or target for treatment.

[0030] The invention is thus also directed to methods using the CPE geneor gene product as a reagent or target to screen for agents thatmodulate the levels or effectively reverse the mutation or otherabnormality in the CPE gene or gene product. Accordingly, the inventionprovides methods for identifying agonists and antagonists of the CPEgene or gene product. These agents can be used to diagnose or treat TypeII diabetes by their effects on the level or function of the CPE gene orgene product. By identifying agents that are capable of modulating theexpression or function of the CPE gene or gene product, methods are thusprovided for affecting the development of or course of Type II diabetesin an individual by modulating the level or function of the CPE gene orgene product. Further, by providing these agents that modulate theexpression, methods are provided for assessing the effect of treatmentin cell and animal models.

[0031] By identifying agents that are capable of interacting with, orotherwise allowing detection of abnormal expression or function of theCPE gene or gene product, methods are thus provided for diagnosing thedevelopment of, or risk of developing, type II diabetes. This can be inthe context of an individual patient, monitoring clinical trials, andassessing CPE gene function or efficacy of treatment in cell and animalmodels.

[0032] The invention further encompasses compositions based on the CPEgene or gene product that are useful for detection or modulation of theexpression or function of the CPE gene or gene product. Thus, thesecompositions are useful for the diagnosis or treatment of Type IIdiabetes.

[0033] The invention also provides cell and animal model systems forstudying Type II diabetes based on alterations in the CPE gene or geneproduct in the model.

[0034] In one embodiment, the polynucleotides and polypeptides useful inthe compositions and methods described herein contain a mutation in thecoding region, arg→trp 283, corresponding to a c→t nucleotide change atthis position. However, any CPE variant that is associated with type IIdiabetes is useful for the compositions and methods described herein.Further, as described below, wild-type CPE gene or gene product can beuseful as a target for treatment and diagnosis in instances in which analteration in CPE that correlates with type II diabetes does not residein the presence of a nucleotide or amino acid mutation.

DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 shows the human carboxypeptidase E amino acid andnucleotide sequence identifying the amino acid change from arginine totryptophan in all members of the family at amino acid 283. Thiscorresponds to a C→T nucleotide change at this position.

[0036]FIG. 2 shows an alignment of carboxypeptidase E from variousspecies (from top to bottom: human, rat, lopam, bovine, murine, andalysia).

[0037]FIG. 3 shows an alignment of carboxypeptidase homologs (CPE, CPM,CPN, and CPZ).

DETAILED DESCRIPTION OF THE INVENTION

[0038] The inventors have discovered that expression of an altered formof carboxypeptidase E is a factor in Type II diabetes in humans.Specifically, the inventors have discovered the occurrence of a specificmutation in the carboxypeptidase E gene in two families with the Type IIdiabetes phenotype. In an initial screening of approximately 20 Type IIdiabetes individuals against 20 controls for mutations in thecarboxypeptidase E gene, the inventors identified a coding mutation(ARG→TRP 283) in an affected individual (FIG. 1). Two affected siblingswere then screened and also found to have the mutation. In this family,all three affected siblings were heterozygous for the mutation.

[0039] Subsequent to this study, the mutation was further identified in3 unrelated affected families. In the first family, three siblings werestudied. The two affected siblings were found to contain the mutation.The mutation was lacking in the third, non-affected sibling. In thesecond affected pedigree, two of the four siblings contained themutation, while the other two did not. The two with the mutationappeared to be most significantly affected, judged by the age of onset.In the third affected family, only one of the two affected siblings hadthis mutation. All affected patients were heterozygous for the mutation.These studies link mis-expression of the carboxypeptidase E gene to typeII diabetes in humans (but not obesity).

[0040] The invention is therefore directed to methods using the CPE geneor gene product as a target to detect Type II diabetes or the risk ofdeveloping Type II diabetes. The invention is also directed to methodsfor determining the molecular basis for type II diabetes or the risk ofdeveloping type II diabetes using the CPE gene or gene product as atarget.

[0041] It is understood that “gene product” refers to all moleculesderived from the gene, especially RNA and protein. cDNA is alsoencompassed, where, for example, made by naturally-occurring reversetranscriptase.

[0042] In one embodiment, the gene itself is detected. Alterations incopy number, genomic position, and nucleotide sequence can be detected.Alterations in nucleotide sequence include insertion, deletion, pointmutation, and inversion. One or more alterations in sequence can occurat any position within the gene, including coding, noncoding,transcribed, and non-transcribed, regulatory regions. Other alterationsthat can be detected include nucleic acid modification, such asmethylation, gross rearrangement in the genome such as in ahomogeneously-staining region, double minute chromosome or otherextrachromosomal element, or cytoskeletal arrangement.

[0043] The invention also encompasses the detection of RNA transcribedfrom the CPE gene. Detection encompasses alterations in copy number andnucleotide sequence. Sequence changes include insertion, deletion, pointmutation, inversion, and splicing variation. Detection of CPE RNA can beindirectly accomplished by means of its cDNA.

[0044] CPE DNA and RNA levels and gross rearrangement can be analyzed byany of the standard methods known in the art. Nucleic acid can beisolated from the cell or analyzed in situ in a cell or tissue sample.For detecting alterations in nucleic acid levels or gross rearrangement,all, or any part, of the nucleic acid molecule can be detected. Nucleicacid reagents derived from any desired region of the CPE gene can beused as a probe or primer for these procedures. Copy number can beassessed by in situ hybridization or isolation of nucleic acid from thecell and quantitation by standard hybridization procedures such asSouthern or Northern analysis. Genes can be amplified in the forms ofhomogeneously-staining regions or double minute chromosomes.Accordingly, one method of detection involves assessing the cellularposition of an amplified gene. This method encompasses standard in situhybridization methods, or alternatively, detection of an amplifiedfragment derived from digestion with an appropriate restriction enzymerecognizing a sequence that is repeated in the amplified unit.

[0045] Identifying nucleic acid modifications, such as methylation, canbe analyzed by any of the known methods in the art for digesting nucleicacid and analyzing modified nucleotides, such as by HPLC, thin-layerchromatography, mass spectra analysis, and the like.

[0046] Gross rearrangements in the genome are preferably detected bymeans of in situ hybridization, although this type of alteration canalso be assessed by means of assays involving normal cellular componentswith which the genes are normally found, such as in specific membranepreparations.

[0047] Mutations in CPE nucleic acid can be analyzed by any of thestandard methods known in the art. Nucleic acid can be isolated from acell or analyzed in situ in a cell or tissue sample by means of specifichybridization probes designed to allow detection of the mutation. Inthis embodiment, the portion of the nucleic acid that is detectedpreferably contains the mutation. However, it is understood that in someembodiments, as where the mutation affects secondary structure or othercellular association, distant regions affected by the mutation can bedetected. In this embodiment, nucleic acid reagents are preferablyderived from the mutated region of the CPE gene to be used as a probe orprimer for the procedures. However, as discussed above, nucleic acidreagents useful as probes can be derived from any position in thenucleic acid. RNA or cDNA can be used in the same way.

[0048] In certain embodiments, detection of the mutation involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al.,PNAS91:360-364 (1994)), the latter of which can be particularly usefulfor detecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0049] Alternatively, mutations in a CPE gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0050] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

[0051] Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature.

[0052] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method.

[0053] Furthermore, sequence differences between a mutant CPE gene and awild-type gene can be determined by direct DNA sequencing. A variety ofautomated sequencing procedures can be utilized when performing thediagnostic assays ((1995) Biotechniques 19:448), including sequencing bymass spectrometry (see, e.g., PCT International Publication No. WO94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffinet al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0054] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include, selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0055] Methods of detection also encompass detection of the CPE protein.Detection encompasses assessing protein levels, mutation,post-translational modification, and subcellular localization. Mutationsencompass deletion, insertion, substitution and inversion. Mutations atRNA splice junctions can result in protein splice variants.

[0056] CPE protein levels can be analyzed by any of the standard methodsknown in the art. Protein can be isolated from the cell or analyzed insitu in a cell or tissue sample. Quantitation can be accomplished insitu, for example by standard of fluorescence detection proceduresinvolving a fluorescently labeled binding partner such as an antibody orother protein with which the CPE protein will bind. This could include asubstrate upon which the protein acts or an enzyme which normally actson the protein. Quantitation of isolated protein can be accomplished byother standard methods for isolated protein, such as in situ geldetection, Western blot, or quantitative protein blot. Levels can alsobe assayed by functional means, such as the effects upon a specificsubstrate. In the case of the CPE protein, this could involve thecleavage of basic amino acids from the C terminus of the various peptidesubstrates upon which the CPE protein normally acts, or artificialsubstrates designed for this assay. It is understood that any enzymeactivity contained in the CPE protein can be used to assess proteinlevels.

[0057] Mutations in CPE protein can be analyzed by any of the above orother standard methods known in the art. Protein can be isolated fromthe cell or analyzed in situ in a cell or tissue sample. Analyticmethods include assays for altered electrophoretic mobility, bindingproperties, tryptic peptide digest, molecular weight, antibody-bindingpattern, isoelectric point, amino acid sequence, and any other of theknown assay techniques useful for detecting mutations in a protein.Assays include, but are not limited to, those discussed in Varlamov etal., J. Biol. Chem. 271:13981 (1996), incorporated herein by referencefor teaching such assays. These include C-terminal arginine binding,acidic pH optima, sensitivity to inhibitors, thermal stability,intracellular distribution, endopeptidase activity, effect onendopeptidase inhibitor, substrate affinity, enzyme kinetics, membraneassociation, posttranslational modification, active site confirmation,compartmentalization, binding to substrate, secretion, and turnover.Further assays for function can be found in Fricker, J. Cell Biochem.38:279-289 (1988), and Manser et al., Biochem. J. 267:517-525, (1990),both incorporated by reference for teaching specific functions that canbe assayed for mutation in the CPE gene.

[0058] In vitro techniques for detection of the protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, the proteincan be detected in vivo in a subject by introducing into the subject alabeled anti-CPE antibody. For example, the antibody can be labeled witha radioactive marker whose presence and location in a subject can bedetected by standard imaging techniques. For detection of specificmutation in the protein, antibodies, or other binding partners, can beused that specifically recognize these alterations. Alternatively,mutations can be detected by direct sequencing of the protein.

[0059] Other alterations that can be detected include alterations inpost-translational modification. Amino acids, including the terminalamino acids, may be modified by natural processes, such as processingand other post-translational modifications. Common modifications thatoccur naturally in polypeptides are described in basic texts, detailedmonographs, and the research literature, and they are well known tothose of skill in the art.

[0060] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0061] Such modifications are well-known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins-Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0062] In addition to detection methods that involve specific physicalfeatures, functional characteristics of the protein are also useful fordetection with known methods. These include changes in biochemistry,such as substrate affinity, enzyme kinetics, membrane association,active site conformation, compartmentalization, forming a complex withsubstrates or enzymes that act upon the protein, secretion, turnover, pHoptima, sensitivity to inhibitors, thermal stability, endopeptidaseactivity, effects on endopeptidase inhibitors, and any other suchfunctional characteristic that is indicative of a mutation or alterationin post-translational modification. Specific assays can be found in theliterature (e.g., see Varlamov et al. (1996) J. Biol. Chem. 271:13981).

[0063] CPE gene and gene product can be detected in a variety ofsystems. These include cell-free and cell-based systems in vitro,tissues, such as ex vivo tissues for returning to a patient, in abiopsy, and in vivo, such as in patients being treated, for monitoringclinical trials, and in animal models. Cell-free systems can be derivedfrom cell lines or cell strains in vitro, including recombinant cells,cells derived from patients, subjects involved in clinical trials, andanimal models, including transgenic animal models. In one embodiment,CPE gene and gene product can also be detected in cell-based systems.This includes cell lines and cell strains in vitro, includingrecombinant lines and strains containing the CPE gene, explanted cellssuch as primary cultures, particularly those derived from a patient withtype II diabetes, subjects undergoing clinical trials, and animal modelsof diabetes including transgenic animals. The CPE gene and gene productcan also be detected in tissues. These include tissues derived frompatients with type II diabetes, subjects undergoing clinical trials, andanimal models. In one embodiment, the tissues are those affected in typeII diabetes. CPE gene and gene product can also be detected inindividual patients with type II diabetes, and subjects undergoingclinical trials, and in animal models of diabetes, including transgenicmodels. Preferred sources of detection include cell and tissue biopsiesfrom individuals affected with diabetes or at risk for developingdiabetes.

[0064] In addition to detecting the CPE gene or gene product directly,the invention also encompasses the use of compounds that produce aspecific effect on a variant CPE gene or gene product as a further meansof diagnosis. This includes, for example, detection of binding partners,including binding partners specific for variant CPE genes or geneproducts, and compounds that have a detectable effect on a function ofCPE genes or gene products. For example, an increase in CPE levels canbe detected by a complex formed between the CPE and a binding partner orlevels of free CPE binding partner. As a further example, abnormallyhigh CPE activity could be detected by concurrently high amounts of CPEprocessed substrate.

[0065] All these methods of detection can be used in procedures toscreen individuals at risk for developing or having Type II diabetes.Further, detection of the alterations of the gene or gene products inindividuals can serve as a prognostic marker for developing diabetes ordiagnostic marker for having diabetes when the individuals are not knownto have diabetes or to be at risk for having diabetes.

[0066] Diagnostic assays can be performed in cell-based systems, andparticularly in cells associated with type II diabetes, in intacttissue, such as a biopsy, and nonhuman animals and humans in vivo.Diagnosis can be at the level of nucleic acid or polypeptide.

[0067] The invention also encompasses methods for modulating the levelor activity of CPE gene or gene product.

[0068] At the level of the gene, known recombinant techniques can beused to alter the gene in vitro or in situ. Excessive copies of, or allor part of, the gene can be deleted. Deletions can be made in anydesired region of the gene including transcribed, non-transcribed,coding and non-coding regions. Additional copies of part or all of thegene can also be introduced into a genome. Finally, alterations innucleotide sequence can be introduced into the gene by recombinanttechniques. Alterations include deletions, insertions, inversions, andpoint mutation. Accordingly, type II diabetes that is caused by amutated CPE gene could be treated by introducing a functional (wildtype) CPE gene into the individual. Further, specific alterations couldbe introduced into the gene and function tested in any given cell type,such as in cell-based models for diabetes. Still further, any givenmutation can be introduced into a cell and used to form a transgenicanimal which can then serve as a model for diabetes and diabetestesting.

[0069] Homologously recombinant host cells can also be produced thatallow the in situ alteration of endogenous CPE polynucleotide sequencesin a host cell genome. This technology is more fully described in WO93/09222, WO 91/12650 and U.S. Pat. No. 5,641,670. Briefly, specificpolynucleotide sequences corresponding to the CPE polynucleotides orsequences proximal or distal to a CPE gene are allowed to integrate intoa host cell genome by homologous recombination where expression of thegene can be affected. In one embodiment, regulatory sequences areintroduced that either increase or decrease expression of an endogenoussequence. Accordingly, a CPE protein can be produced in a cell notnormally producing it, or increased expression of CPE protein can resultin a cell normally producing the protein at a specific level.

[0070] The levels and activity of CPE RNA are also subject tomodulation. Polynucleotides corresponding to any desired region of theRNA can be used directly to block transcription or translation of CPEsequences by means of antisense or ribozyme constructs. Thus, where thedisorder is characterized by abnormally high gene expression, thesenucleic acids can be used to decrease expression levels. A DNA antisensepolynucleotide is designed to be complementary to a region of the geneinvolved in transcription, preventing transcription and hence productionof protein. An antisense RNA or DNA polynucleotide would hybridize tothe mRNA and thus block translation of mRNA into protein. An alternativetechnique involves cleavage by ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated.

[0071] The invention also encompasses the modulation of nucleic acidexpression using compounds that have been discovered by screening theeffects of the compounds on CPE nucleic acid levels or function.

[0072] The invention is further directed to methods for modulating CPEprotein levels or function. For example, where the disorder ischaracterized by an overexpression of the protein, antibodies or otherbinding partners directed against the protein can be used to block theprotein function and thus functionally decrease the levels of proteinpresent. Antibodies can be prepared against specific fragmentscontaining sites required for function or against the intact protein.Protein levels can also be modulated by use of compounds discovered inscreening techniques in which the protein levels serve as a target foreffective compounds. Finally, mutant proteins can be functionallyaffected by the use of compounds discovered in screening techniques thatuse an alteration of mutant function as an end point.

[0073] Modulation can be in a cell-free system. In this context, forexample, the assay could involve cleavage of substrate or otherindicator of CPE activity. Modulation can also occur in cell-basedsystems. These cells may be permanent cell lines, cell strains, primarycultures, recombinant cells, cells derived from affected individuals,and transgenic animal models of diabetes, among others. Modulation canalso be in vivo, for example, in patients having the disorder, insubjects undergoing clinical trials, and animal models of diabetes,including transgenic animal models. Modulation could be measured bydirect assay of the CPE gene or gene product or by the results of CPEgene and gene product function, for example, insulin responsiveness,hepatic glucose production, insulin secretion, pro-insulin levels, andany of the functional indicators described herein and known to those ofordinary skill in the art.

[0074] All of these methods can be used to affect CPE function inindividuals having or at risk for having Type II diabetes. Thus, theinvention encompasses the treatment of Type II diabetes by modulatingthe levels or function of CPE genes or gene product.

[0075] The invention also encompasses methods for identifying compoundsthat interact with the CPE gene or gene product, particularly tomodulate the level or function of the CPE gene or gene product.Modulation can be at the level of transcription, translation, orpolypeptide function. Accordingly, where levels of CPE gene or geneproduct are abnormally high or low, compounds can be screened for theability to correct the level of expression. Alternatively, where amutation affects the function of the CPE nucleic acid or protein,compounds can be screened for their ability to compensate for or tocorrect the dysfunction. In this manner, CPE and CPE variants can beused to identify agonists and antagonists useful for affecting CPE andvariant gene expression. These compounds can then be used to affect CPEexpression or function in Type II diabetes. Thus these screening methodsare useful to identify compounds that can be used for treating Type IIdiabetes.

[0076] These compounds are also useful in a diagnostic context in thatthey can then be used to identify altered levels of CPE or CPE variantsin a cell, tissue, nonhuman animal, and human. For example, compoundsspecifically interacting with CPE nucleic acid or protein to produce aparticular result, by producing that result in a cell, tissue, nonhumananimal, or human, indicate that there is a lesion in the CPE gene orgene product.

[0077] Thus, modulators of gene expression can be identified in a methodwherein CPE gene or gene product is contacted with a candidate compoundand the level or expression of gene or gene product is determined. Thelevel or expression of gene or gene product in the presence of thecandidate compound is compared to the level or expression of gene orgene product in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of nucleic acid orprotein expression based on this comparison and be used, for example, totreat type II diabetes. When the level or expression of gene or geneproduct is statistically significantly greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of levels or expression of the gene or geneproduct. When levels or product expression are statisticallysignificantly less in the presence of the candidate compound than in itsabsence, the candidate compound is identified as an inhibitor.

[0078] These compounds can be used to test on model systems, includinganimal models of diabetes, and human clinical trial subjects, cellsderived from these sources as well as transgenic animal models ofdiabetes.

[0079] Accordingly, the invention provides methods of treatment, withthe gene or gene product as a target, using a compound identifiedthrough drug screening as a modulator to modulate expression of the geneor gene product. Modulation includes both up-regulation (i.e. activationor agonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

[0080] Further, the expression of genes that are up- or down-regulatedin response to CPE can also be assayed. In this embodiment theregulatory regions of these genes can be operably linked to a reportergene such as luciferase.

[0081] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0082] Other candidate compounds include preprocessed peptides that arenormal substrates for CPE, for example, enkephalin, insulin, andproneurotensin, i.e., peptides treated so that the amino terminal basicresidues have been cleaved.

[0083] Any of the biological or biochemical functions mediated by CPEcan be used in an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in theart.

[0084] A further aspect of the invention involves pharmacogenomicanalysis in the case of polymorphic CPE proteins and specific mutants.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M., Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996), and Linder, M. W., Clin.Chem. 43(2):254-266 (1997). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. Accordingly, in one aspect of the invention, natural variantsof the CPE protein are used to screen for compounds that are effectiveagainst a given allele and are not toxic to the specific patient.Compounds can thus be classed according to their effects againstnaturally occurring allelic variants. This allows more effectivetreatment and diagnosis of type II diabetes.

[0085] Test systems for identifying compounds include both cell-free andcell-based systems derived from normal and affected tissue, cell linesand strains, primary cultures, animal diabetes models, and includingtransgenic animals. Naturally-occurring cells will express abnormallevels of CPE gene or gene product or variants of CPE genes or geneproducts. Alternatively, these cells can provide recombinant hosts forthe expression of desired levels of CPE gene or gene product or variantsof CPE gene or gene product. A cell-free system can be used, forexample, when assessing the effective agents on nucleic acid orpolypeptide function.

[0086] For example, in a cell-free system, competition binding assaysare designed to discover compounds that interact with the polypeptide.Thus, a compound is exposed to the polypeptide under conditions thatallow the compound to bind or to otherwise interact with thepolypeptide. Soluble polypeptide is also added to the mixture. If thetest compound interacts with the soluble polypeptide, it decreases theamount of complex formed or activity from the target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the polypeptide. Thus, the solublepolypeptide that competes with the target region is designed to containpeptide sequences corresponding to the region of interest. In oneembodiment, the region of interest is the 51 C-terminal amino acids thatdirect sorting to the membrane. Another region is that located around23-33 amino acids from the C-terminal region, required for properfolding. A third region is located within the predicted amphiphilichelix of the C-terminal 14 amino acid residues, involved in binding CPEto membranes.

[0087] To perform cell-free drug screening assays, it is desirable toimmobilize either the protein, or fragment, or its target molecule tofacilitate separation of complexes from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.

[0088] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/CPE fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of CPE-binding protein found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques. For example,either the polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin using techniques wellknown in the art. Alternatively, antibodies reactive with the proteinbut which do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and the proteintrapped in the wells by antibody conjugation. Preparations of aCPE-binding protein and a candidate compound are incubated in the CPEprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the CPEprotein target molecule, or which are reactive with the CPE protein andcompete with the target molecule; as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetmolecule.

[0089] Cell-based systems include assay of individual cells or assay ofcells in a tissue sample or in vivo. Drug screening assays can becell-based or cell-free systems. Cell-based systems can be native, i.e.,cells that normally express the protein, as a biopsy or expanded in cellculture. In one embodiment, however, cell-based assays involverecombinant host cells expressing the protein. In vivo test systemsinclude, not only individuals involved in clinical trials, but alsoanimal diabetes models, including transgenic animals. Single cellsinclude recombinant host cells in which desired altered CPE gene or geneproducts have been introduced. These host cells can express abnormallyhigh or low levels of the CPE gene or gene product or mutant versions ofthe CPE gene or gene product. Thus, the recombinant cells can be used astest systems for identifying compounds that have the desired effect onthe altered gene or gene product. Mutations can be naturally occurringor constructed for their effect on the course or development of type IIdiabetes, for example, determined by the model test systems discussedfurther below. Similarly, naturally-occurring or designed mutations canbe introduced into transgenic animals, which then serve as an in vivotest system to identify compounds having a desired effect on CPE gene orgene product.

[0090] In yet another aspect of the invention, the polypeptides can beused in a “two hybrid” assay (see, for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), for isolating coding sequences forother cellular proteins which bind to or interact with CPE.

[0091] Briefly, the two hybrid assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for a CPE polyp eptide.The second hybrid protein encodes a transcriptional activation domainfused in frame to a sample gene from a cDNA library. If the bait andsample hybrid proteins are able to interact, e.g., form a CPE-dependentcomplex, they bring into close proximity the two domains of thetranscriptional activator. This proximity is sufficient to causetranscription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the reporter gene can be detected and usedto score for the interaction of CPE and sample proteins.

[0092] Modulators of CPE gene or gene product identified according tothese assays can be used to treat type II diabetes by treating cellsthat aberrantly express the gene or gene product. These methods oftreatment include the steps of administering the modulators of proteinactivity in a pharmaceutical composition as described herein, to asubject in need of such treatment.

[0093] The invention thus provides a method for identifying a compoundthat can be used to treat type II diabetes. The method typicallyincludes assaying the ability of the compound to modulate the expressionof the CPE gene or gene product to identify a compound that can be usedto treat the disorder.

[0094] The invention is also directed to CPE genes or gene productscontaining alterations that correlate with Type II diabetes. Thesealtered genes or gene products can be isolated and purified or can becreated in situ, for example, by means of in situ gene replacementtechniques. In the gene, alterations of this type can be found in anysite, transcribed, nontranscribed, coding, and noncoding. Likewise, inthe RNA, alterations can be found in both the coding and noncodingregions. In a specific disclosed embodiment, the invention encompasses acoding mutation, arg→trp 283, corresponding to a c→t nucleotide changeat this position. In one embodiment, the carboxypeptidase E gene or geneproduct comprises a fragment, preferably a fragment containing themutation. The invention thus encompasses primers, both wild type andvariant, that are useful in the methods described herein. Similarly,ribozymes and antisense nucleic acids can be derived from variants thatcorrelate with type II diabetes or can be derived from the wild type andused in the methods described herein.

[0095] The genes and gene products are useful in pharmaceuticalcompositions for diagnosing or modulating the level or expression of CPEgene or gene product in vivo, as in individual patients treated for typeII diabetes, subjects in clinical trials, animal diabetes models, andtransgenic animal diabetes models. Thus, these pharmaceuticalcompositions are useful for testing and treatment. The CPE genes or geneproducts are also useful for otherwise modulating expression of the geneor gene product in cell-free or cell-based systems in vitro. They arefurther useful in ex vivo applications. The genes and gene products arealso useful for creating model test systems for type II diabetes, forexample, recombinant cells, tissues, and animals. The genes and geneproducts are also useful in a diagnostic context as comparisons forother naturally-occurring variation in the CPE gene or gene product.Accordingly, these reagents can form the basis for a diagnostic kit.Further, specific variants (mutants) are useful for testing compoundsthat may be effective in the treatment or diagnosis of type II diabetes.Such mutants can also form the basis of a reagent in a test kit,particularly for introduction into a desired cell type or transgenicanimal for drug testing. Accordingly, the invention is also directed toisolated and purified polypeptides and polynucleotides.

[0096] The invention is thus directed to compositions based oncarboxypeptidase E genes or gene products. Compositions also includenucleic acid primers derived from carboxypeptidase E mutants, antisensenucleotides derived from these mutants, and ribozymes based on themutations, and antibodies specific for the mutants. Compositions furtherinclude recombinant cells containing any of the mutants, vectorscontaining the mutants, cells expressing the mutants, fragments of themutants, and antibodies or other binding partners that specificallyrecognize the mutation. These compositions can all be combined with apharmaceutically acceptable carrier to create pharmaceuticalcompositions useful for detecting or modulating the level or expressionof carboxypeptidase E gene or gene product and thereby diagnosing ortreating Type II diabetes.

[0097] As used herein, a polypeptide is said to be “isolated” or“purified” when it is substantially free of cellular material when it isisolated from recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can be joined to another polypeptide with which itis not normally associated in a cell and still be considered “isolated”or “purified.”

[0098] The CPE polypeptides can be purified to homogeneity. It isunderstood, however, that preparations in which the polypeptide is notpurified to homogeneity are useful and considered to contain an isolatedform of the polypeptide. The critical feature is that the preparationallows for the desired function of the polypeptide, even in the presenceof considerable amounts of other components. Thus, the inventionencompasses various degrees of purity.

[0099] In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the polypeptide having less thanabout 30% (by dry weight) other proteins (i.e., contaminating protein),less than about 20% other proteins, less than about 10% other proteins,or less than about 5% other proteins. When the CPE polypeptide isrecombinantly produced, it can also be substantially free of culturemedium, i.e., culture medium represents less than about 20%, less thanabout 10%, or less than about 5% of the volume of the proteinpreparation.

[0100] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the polypeptide in which it isseparated from chemical precursors or other chemicals that are involvedin its synthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thepolypeptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0101] Variants can be naturally-occurring or can be made by recombinantmeans or chemical synthesis to provide useful and novel characteristicsfor the polypeptide. This includes preventing immunogenicity frompharmaceutical formulations by preventing protein aggregation.

[0102] Useful variations further include alteration of bindingcharacteristics. For example, one embodiment involves a variation at thebinding site that results in binding but not release, or slower release,of substrate. A further useful variation at the same sites can result ina higher affinity for substrate. Useful variations also include changesthat provide for affinity for another substrate. Another usefulvariation includes one that allows binding but which reduces cleavage ofthe substrate.

[0103] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)). The latter procedure introduces single alanine mutations atevery residue in the molecule. The resulting mutant molecules are thentested for biological activity. Sites that are critical can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0104] The invention also provides antibodies that selectively bind tothe protein. An antibody is considered to selectively bind, even if italso binds to other proteins that are not substantially homologous withthe protein. These other proteins share homology with a fragment ordomain of the protein. This conservation in specific regions gives riseto antibodies that bind to both proteins by virtue of the homologoussequence. In this case, it would be understood that antibody binding tothe CPE protein is still selective.

[0105] To generate antibodies, an isolated polypeptide is used as animmunogen to generate antibodies using standard techniques forpolyclonal and monoclonal antibody preparation. Either the full-lengthprotein or antigenic peptide fragment can be used.

[0106] Antibodies are preferably prepared from these regions or fromdiscrete fragments in these regions. However, antibodies can be preparedfrom any region of the peptide as described herein. A preferred fragmentproduces an antibody that diminishes or completely preventssubstrate-binding. Antibodies can be developed against the entireprotein or portions of the protein, for example, the substrate bindingdomain.

[0107] Antibodies can be polyclonal or monoclonal. An intact antibody,or a fragment thereof (e.g. Fab or F(ab′)₂) can be used.

[0108] Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0109] An appropriate immunogenic preparation can be derived fromnative, recombinantly expressed, protein or chemically synthesizedpeptides.

[0110] The antibodies can be used to isolate a CPE protein by standardtechniques, such as affinity chromatography or immunoprecipitation. Theantibodies can facilitate the purification of the natural protein fromcells and recombinantly-produced protein expressed in host cells.

[0111] The antibodies are useful to detect the presence of protein incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism.

[0112] The antibodies can be used to detect the protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression.

[0113] The antibodies can be used to assess abnormal tissue distributionor abnormal expression during development.

[0114] Antibody detection of circulating fragments of the full lengthCPE protein can be used to identify CPE turnover.

[0115] Further, the antibodies can be used to assess CPE expression inactive stages of diabetes or in an individual with a predispositiontoward diabetes. When the disorder is caused by an inappropriate tissuedistribution, developmental expression, or level of expression of theCPE protein, the antibody can be prepared against the normal CPEprotein. If a disorder is characterized by a specific mutation in theCPE protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant CPE protein. However,intracellularly-made antibodies (“intrabodies”) are also encompassed,which would recognize intracellular CPE peptide regions.

[0116] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Antibodies can be developed against the whole CPE or portions of theCPE.

[0117] The diagnostic uses can be applied, not only in genetic testing,but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting CPE expression level or thepresence of aberrant CPE and aberrant tissue distribution ordevelopmental expression, antibodies directed against the CPE orrelevant fragments can be used to monitor therapeutic efficacy.

[0118] The antibodies are also useful for inhibiting CPE function. Theseuses can also be applied in a therapeutic context. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact CPE associated with a cell.

[0119] An “isolated” CPE nucleic acid is one that is separated fromother nucleic acid present in the natural source of the CPE nucleicacid. Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. However, there can be some flankingnucleotide sequences, for example up to about 5 KB. The important pointis that the nucleic acid is isolated from flanking sequences such thatit can be subjected to the specific manipulations described herein suchas recombinant expression, preparation of probes and primers, and otheruses specific to the nucleic acid sequences.

[0120] Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0121] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0122] The CPE polynucleotides can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0123] The CPE polynucleotides include, but are not limited to, thesequence encoding the mature polypeptide alone, the sequence encodingthe mature polypeptide and additional coding sequences, such as a leaderor secretory sequence (e.g., a pre-pro or pro-protein sequence), thesequence encoding the mature polypeptide, with or without the additionalcoding sequences, plus additional non-coding sequences, for exampleintrons and non-coding 5′ and 3′ sequences such as transcribed butnon-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the polynucleotide may befused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

[0124] Polynucleotides can be in the form of RNA, such as mRNA, or inthe form DNA, including cDNA and genomic DNA obtained by cloning orproduced by chemical synthetic techniques or by a combination thereof.The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0125] The invention also provides CPE nucleic acid molecules encodingthe variant polypeptides described herein. Such polynucleotides may benaturally-occurring, such as allelic variants (same locus), homologs(different locus), and orthologs (different organism), or may beconstructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to polynucleotides, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions.

[0126] Variation can occur in either or both the coding and non-codingregions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0127] Furthermore, the invention provides polynucleotides that comprisea fragment of the full length CPE polynucleotides. The fragment can besingle or double stranded and can comprise DNA or RNA. The fragment canbe derived from either the coding or the non-coding sequence.

[0128] The invention also provides CPE nucleic acid fragments thatencode epitope bearing regions of the CPE proteins described herein.

[0129] The invention also provides vectors containing the CPEpolynucleotides. The term “vector” refers to a vehicle, preferably anucleic acid molecule, that can transport the CPE polynucleotides. Whenthe vector is a nucleic acid molecule, the CPE polynucleotides arecovalently linked to the vector nucleic acid. With this aspect of theinvention, the vector includes a plasmid, single or double strandedphage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0130] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the CPE polynucleotides. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of theCPE polynucleotides when the host cell replicates.

[0131] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the CPEpolynucleotides. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0132] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the CPE polynucleotides such thattranscription of the polynucleotides is allowed in a host cell. Thepolynucleotides can be introduced into the host cell with a separatepolynucleotide capable of affecting transcription. Thus, the secondpolynucleotide may provide a trans-acting factor interacting with thecis-regulatory control region to allow transcription of the CPEpolynucleotides from the vector. Alternatively, a trans-acting factormay be supplied by the host cell. Finally, a trans-acting factor can beproduced from the vector itself.

[0133] It is understood, however, that in some embodiments,transcription and/or translation of the CPE polynucleotides can occur ina cell free system.

[0134] The regulatory sequence to which the polynucleotides describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

[0135] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0136] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0137] A variety of expression vectors can be used to express a CPEpolynucleotide. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0138] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0139] The CPE polynucleotides can be inserted into the vector nucleicacid by well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0140] The vector containing the appropriate polynucleotide can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0141] As described herein, it may be desirable to express thepolypeptide as a fusion protein. Accordingly, the invention providesfusion vectors that allow for the production of the CPE polypeptides.Fusion vectors can increase the expression of a recombinant protein,increase the solubility of the recombinant protein, and aid in thepurification of the protein by acting for example as a ligand foraffinity purification. A proteolytic cleavage site may be introduced atthe junction of the fusion moiety so that the desired polypeptide canultimately be separated from the fusion moiety. Proteolytic enzymesinclude, but are not limited to, factor Xa, thrombin, and enterokinase.Typical fusion expression vectors include pGEX (Smith et al., Gene67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein. Examples of suitable inducible non-fusion E.coli expression vectors include pTrc (Amann et al., Gene 69:301-315(1988)) and pET 11 d (Studier et al., Gene Expression Technology:Methods in Enzymology 185:60-89 (1990)).

[0142] Recombinant protein expression can be maximized in a hostbacteria by providing a genetic background wherein the host cell has animpaired capacity to proteolytically cleave the recombinant protein.(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990)119-128). Alternatively, thesequence of the polynucleotide of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0143] The CPE polynucleotides can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0144] The CPE polynucleotides can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0145] In certain embodiments of the invention, the polynucleotidesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

[0146] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the CPEpolynucleotides. The person of ordinary skill in the art would be awareof other vectors suitable for maintenance propagation or expression ofthe polynucleotides described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0147] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0148] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0149] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0150] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the CPE polynucleotides can be introduced either aloneor with other polynucleotides that are not related to the CPEpolynucleotides such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe CPE polynucleotide vector.

[0151] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0152] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the polynucleotides described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0153] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0154] Where secretion of the polypeptide is desired, appropriatesecretion signals are incorporated into the vector. The signal sequencecan be endogenous to the CPE polypeptides or heterologous to thesepolypeptides.

[0155] Where the polypeptide is not secreted into the medium, theprotein can be isolated from the host cell by standard disruptionprocedures, including freeze thaw, sonication, mechanical disruption,use of lysing agents and the like. The polypeptide can then be recoveredand purified by well-known purification methods including ammoniumsulfate precipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

[0156] It is also understood that depending upon the host cell inrecombinant production of the polypeptides described herein, thepolypeptides can have various glycosylation patterns, depending upon thecell, or maybe non-glycosylated as when produced in bacteria. Inaddition, the polypeptides may include an initial modified methionine insome cases as a result of a host-mediated process.

[0157] The host cells expressing the polypeptides described herein, andparticularly recombinant host cells, have a variety of uses. First, thecells are useful for producing CPE proteins or polypeptides that can befurther purified to produce desired amounts of CPE protein or fragments.Thus, host cells containing expression vectors are useful forpolypeptide production.

[0158] Host cells are also useful for conducting cell based assaysinvolving the CPE or CPE fragments. Thus, a recombinant host cellexpressing a native CPE is useful to assay for compounds that stimulateor inhibit CPE function.

[0159] Host cells are also useful for identifying CPE mutants in whichthese functions are affected. If the mutants naturally occur, host cellscontaining the mutations are useful to assay compounds that have adesired effect on the mutant CPE (for example, stimulating or inhibitingfunction) which may not be indicated by their effect on the native CPE.

[0160] Recombinant host cells are also useful for expressing thechimeric polypeptides described herein to assess compounds that activateor suppress activation by means of a heterologous amino terminalextracellular domain (or other binding region). Alternatively, aheterologous region spanning the entire transmembrane domain (or partsthereof) can be used to assess the effect of a desired amino terminalextracellular domain (or other binding region) on any given host cell.In this embodiment, a region spanning the entire transmembrane domain(or parts thereof) compatible with the specific host cell is used tomake the chimeric vector. Alternatively, a heterologous carboxy terminalintracellular, e.g., signal transduction, domain can be introduced intothe host cell.

[0161] Further, mutant CPEs can be designed in which one or more of thevarious functions is engineered to be increased or decreased used toaugment or replace CPE proteins in an individual. Thus, host cells canprovide a therapeutic benefit by replacing an aberrant CPE or providingan aberrant CPE that provides a therapeutic result. In one embodiment,the cells provide CPE that is abnormally active.

[0162] Homologously recombinant host cells can also be produced thatallow the in situ alteration of endogenous CPE polynucleotide sequencesin a host cell genome. This technology is more fully described in WO93/09222, WO 91/12650 and U.S. Pat. No. 5,641,670. Briefly, specificpolynucleotide sequences corresponding to the CPE polynucleotides orsequences proximal or distal to a CPE gene are allowed to integrate intoa host cell genome by homologous recombination where expression of thegene can be affected. In one embodiment, regulatory sequences areintroduced that either increase or decrease expression of an endogenoussequence. Accordingly, a CPE protein can be produced in a cell notnormally producing it, or increased expression of CPE protein can resultin a cell normally producing the protein at a specific level.

[0163] In one embodiment, the host cell can be a fertilized oocyte orembryonic stem cell that can be used to produce a transgenic animalcontaining the altered CPE gene. Alternatively, the host cell can be astem cell or other early tissue precursor that gives rise to a specificsubset of cells and can be used to produce transgenic tissues in ananimal. See also Thomas et al., Cell 51:503 (1987) for a description ofhomologous recombination vectors. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous CPEgene is selected (see e.g., Li, E. et al., Cell 69:915 (1992)). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos. WO 90/11354; WO91/01140; and WO 93/04169.

[0164] The genetically engineered host cells can be used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a CPE protein andidentifying and evaluating modulators of CPE protein activity.

[0165] Other examples of transgenic animals include non-human primates,sheep, dogs, cows, goats, chickens, and amphibians.

[0166] In one embodiment, a host cell is a fertilized oocyte or anembryonic stem cell into which CPE polynucleotide sequences have beenintroduced.

[0167] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the CPE nucleotide sequencesdescribed herein, especially the altered sequences, can be introduced asa transgene into the genome of a non-human animal, such as a mouse.

[0168] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the CPE protein to particular cells.

[0169] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0170] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0171] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0172] Transgenic animals containing recombinant cells that express thepolypeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,CPE activation, and signal transduction, may not be evident from invitro cell free or cell based assays. Accordingly, it is useful toprovide non-human transgenic animals to assay in vivo CPE function, theeffect of specific mutant CPEs on CPE function, and the effect ofchimeric CPEs. It is also possible to assess the effect of nullmutations, that is mutations that substantially or completely eliminateone or more CPE functions.

[0173] The CPE nucleic acid molecules, protein (particularly fragments,such as the domains that interact with other cellular components),modulators of the nucleic acid and protein, and especially bindingpartners, and antibodies (also referred to herein as “active compounds”)can be incorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallycomprise the nucleic acid molecule, protein, modulator, or antibody anda pharmaceutically acceptable carrier.

[0174] As used herein the language “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

[0175] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0176] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a CPE protein or anti-CPE antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0177] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0178] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0179] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0180] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0181] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0182] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated, each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0183] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see e.g., Chen et al., PNAS 91:3054-3057 (1994)). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

[0184] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0185] This invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will fully conveythe invention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

That which is claimed:
 1. A method for detecting a mutation in the CPEgene or gene product in a human subject having, or predisposed tohaving, Type II diabetes, said method comprising: (a) obtaining abiological sample from a subject having, or predisposed to having, TypeII diabetes, said sample containing a CPE gene or gene product; and (b)detecting an alteration in said CPE gene or gene product by means of anassay that allows detection of said alteration in combination with anincrease or decrease in the level of carboxypeptidase E gene expressionin a patient compared to a control subject.
 2. A method of diagnosingType II diabetes or predisposition to having Type II diabetes in a humansubject, said method comprising: (a) obtaining a biological sample fromsaid subject, said sample containing carboxypeptidase E gene or geneproduct; (b) identifying an alteration in carboxypeptidase E gene orgene product by means of an assay that allows detection of saidalteration in combination with an increase or decrease in the level ofcarboxypeptidase E gene expression in a patient compared to a controlsubject; and (c) correlating said alteration with Type II diabetes orthe predisposition for developing Type II diabetes in said subject.
 3. Amethod for determining whether a human subject has or is at risk fordeveloping type II diabetes comprising the steps of: (a) obtaining asample from a subject, said sample comprising nucleic acid moleculescontaining a carboxypeptidase E (CPE) gene; and (b) detecting thepresence or absence of a genetic mutation in the gene of said subject,wherein said genetic mutation comprises an alteration in the codon whichcodes for amino acid 283 which results in a replacement of arginine byanother amino acid and the presence of said genetic mutation identifiesa subject that has or is at risk for developing type II diabetes.
 4. Themethod according to claim 3 , wherein the arginine is replaced by theamino acid tryptophan.
 5. A method for determining whether a humansubject has or is at risk for developing type II diabetes comprising thesteps of: (a) obtaining a sample from a subject, said sample comprisingnucleic acid molecules containing a carboxypeptidas E (CPE) gene; and(b) detecting the presence or absence of a genetic mutation in the geneof said subject, wherein said genetic mutation comprises an alterationin the codon beginning at nucleotide 1133, which results in areplacement of arginine by another amino acid.
 6. The method accordingto claim 4 , wherein the arginine is replaced by the amino acidtryptophan.
 7. A method for determining whether a subject has or is atrisk for developing type II diabetes comprising the steps of: (a)obtaining a sample from a subject, said sample comprising nucleic acidmolecules containing a carboxypeptidas E (CPE) gene; and (b) determiningthe copy number of said CPE gene compared to a control subject.
 8. Amethod for determining whether a subject has or is at risk fordeveloping type II diabetes comprising the steps of: (a) obtaining asample from a subject, said sample comprising nucleic acid moleculescontaining a carboxypeptidas E (CPE) gene; and (b) identifying analteration in carboxypeptidase E (CPE) gene by means of an assay thatallows detection of an alteration in said gene wherein said alterationis selected from the group consisting of: an insertion, a deletion, apoint mutation, and an inversion in said gene.
 9. A method fordetermining whether a subject has or is at risk for developing type IIdiabetes comprising the steps of: (a) obtaining a sample from a subject,said sample comprising nucleic acid molecules containing acarboxypeptidase E (CPE) gene; and (b) identifying a gross rearrangementof said CPE gene in the genome of said subject using in situhybridization.